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Protein nebulization
~ Pergamon
PIT: 50021-8502(96)00188-7
J. A.,• .toI sa; Vol. 27. 5uppl. I. pp . 5231-5232. 1996 Copyright 01996 Elsevier Science LId Prinled i. Oreat Britai• . All righll rcterVCd 0021·8502/96 S!S.OO + 0.00
PROTEIN NEBULIZATION ••
I
2
I. FANGMARK and J. C. CARPIN , I National Defence Research Establishment, S-901 82 Umea, Sweden 2 U S Army, Edgewood Research Development and Engineering Center, Aberdeen Proving Ground, MD 21010-5423, USA
KEYWORDS Aerosolization, Inhalation aerosol. Protein Stability. ImmunoglobulineG, Urease
INTRODUCTION The use of aerosols for inhalation exposure studies and therapeutic purposes requires the production of an intact and fully active aerosol with well-characterized properties. Special precautions must therefore be taken when aerosolizing biologically active substances, such as proteins, since exposure to the disintegrating forces during the production of aerosols may alter their biological activity. Shear forces, surface denaturation at the hydrophobic air-water interface or enhanced chemical reaction rates due to the huge increase in total surface area produced has been suggested as possible degradation mechanisms (Niven, 1994). This study was undertaken in order to reach a better understanding about which aerosolization parameters that are important for the degradation of sensitive substances during aerosolization. METHODS The effects of the liquid feed rate, the gauge pressure. the atmosphere. the relative humidity (RH) and the number of recirculations on the activity of proteins after aerosolization were quantified based on an experime~tal pI~?f a two-level full factorial desig~. Gauge . pressures in the range 25 - 35 PSI an~ l~qUld ~eed rates between 2 - 18 ml/mm were investigated. Dry (<5 % RH) or humidified air (70 % RH) or dry or humidified N2' supphed from a gas cylinder. were used for aerosolization. The experiments were performed with a Constant Output Atomizer (TSI 3076) operated in the non-recirculating mode. Solutions of proteins in phosphate buffered saline (PBS) were aerosolized and the drained liquid collected and analyzed for activity and total protein content. Two proteins were chosen for this study: the enzyme urease and an antibody. rabbitImmunoglobulinG. (IgG). The activity of urease was measured photometrically as the rate constant of the enzymatic degradation of urea. Enzyme linked immunosorbent assay (ELISA) was used to study the acti~ity of IgG. The total protein concentration was estimated from the cr content of the PBS by Ion chromatographic analysis. The liquid was further characterized by spectrophotometric measurements in the visible and ultraviolet wavelength range. Some additional tests were performed with a 3-jet Collison nebulizer (May. 1973) to assess the practical implications of the former experiments. In this case the time course of protein 8231
S232
Abstracts of the 1996 European Aerosol Conference
degradation was studied. Samples of protein solutions were withdrawn from the liquid reservoir at different times and analyzed as previously described. RESULTS Importance of aerosolization parameters The results show that the most important parameters for the degradation of urease is the liquid flow rate, followed by the relative humidity of the atmosphere (Fangmark and Carpin, 1996). Degradation occurred both during atomization with compressed air and with N2, showing that oxidation is not a primary degradation pathway. Within the experimental range, 1- 9 recirculations, the number of recirculations was found to be the dominating parameter for the degradation of IgG, followed by the relative humidity of the atmosphere and the liquid feed rate. Thus deactivation is likely to occur at the airlliquid interface where shear forces and water evaporation may act to bring reactants closer together and produce a local environment with a high ion strength. The results also imply that denaturation is caused by aggregation. Time course of degradation Two types of deactivation behavior were observed with a Collison nebulizer. The first type, represented by urease, is a linear degradation with time on a logarithmic scale (Type I). The other type, represented by IgG has a rapid initial degradation rate that levels-off after approximately 5 - 10 minutes of nebulization (Type II). Accordingly an equal fraction of urease is degraded by surface forces during each passage through the nebulizer. For IgG, the time course of degradation correlates well with the temperature decrease of the nebulizer solution. Thus, for IgG, effects caused by the evaporation of water may be the most important. In earlier studies on the time course of protein degradation, Type I degradation was observed for the enzyme lactase-dehydrogenase while the protein recombinant granulocyte colony stimulating factor follow type II degradation (Niven et al, 1994).
ACKNOWLEDGEMENTS Support from an ERDEC-National Research Council Research Associateship award is gratefully acknowledged. REFERENCES Fangmark I., and Carpin, lC. Stability of proteins during aerosolization. The effect of operating conditions on urease enzymatic activity. Submitted to AerosolSci Techn. May, K.R. (1973). The Collison nebulizer: Description, Performance and Application.
Aerosol Sci, 4,235 - 243. Niven, R. W, Ip, A. Y, Mittelman, S., Farrar, C., Arakawa, T, and S. J. Prestrelski. (1994) . Protein nebulization: I. Stability of lactate dehydrogenase and recombinant granulocytecolony stimulating factor to air-jet nebulization. Int J Pharm 109, 17 - 26.
PIT: 50021-8502(96)00188-7
J. A.,• .toI sa; Vol. 27. 5uppl. I. pp . 5231-5232. 1996 Copyright 01996 Elsevier Science LId Prinled i. Oreat Britai• . All righll rcterVCd 0021·8502/96 S!S.OO + 0.00
PROTEIN NEBULIZATION ••
I
2
I. FANGMARK and J. C. CARPIN , I National Defence Research Establishment, S-901 82 Umea, Sweden 2 U S Army, Edgewood Research Development and Engineering Center, Aberdeen Proving Ground, MD 21010-5423, USA
KEYWORDS Aerosolization, Inhalation aerosol. Protein Stability. ImmunoglobulineG, Urease
INTRODUCTION The use of aerosols for inhalation exposure studies and therapeutic purposes requires the production of an intact and fully active aerosol with well-characterized properties. Special precautions must therefore be taken when aerosolizing biologically active substances, such as proteins, since exposure to the disintegrating forces during the production of aerosols may alter their biological activity. Shear forces, surface denaturation at the hydrophobic air-water interface or enhanced chemical reaction rates due to the huge increase in total surface area produced has been suggested as possible degradation mechanisms (Niven, 1994). This study was undertaken in order to reach a better understanding about which aerosolization parameters that are important for the degradation of sensitive substances during aerosolization. METHODS The effects of the liquid feed rate, the gauge pressure. the atmosphere. the relative humidity (RH) and the number of recirculations on the activity of proteins after aerosolization were quantified based on an experime~tal pI~?f a two-level full factorial desig~. Gauge . pressures in the range 25 - 35 PSI an~ l~qUld ~eed rates between 2 - 18 ml/mm were investigated. Dry (<5 % RH) or humidified air (70 % RH) or dry or humidified N2' supphed from a gas cylinder. were used for aerosolization. The experiments were performed with a Constant Output Atomizer (TSI 3076) operated in the non-recirculating mode. Solutions of proteins in phosphate buffered saline (PBS) were aerosolized and the drained liquid collected and analyzed for activity and total protein content. Two proteins were chosen for this study: the enzyme urease and an antibody. rabbitImmunoglobulinG. (IgG). The activity of urease was measured photometrically as the rate constant of the enzymatic degradation of urea. Enzyme linked immunosorbent assay (ELISA) was used to study the acti~ity of IgG. The total protein concentration was estimated from the cr content of the PBS by Ion chromatographic analysis. The liquid was further characterized by spectrophotometric measurements in the visible and ultraviolet wavelength range. Some additional tests were performed with a 3-jet Collison nebulizer (May. 1973) to assess the practical implications of the former experiments. In this case the time course of protein 8231
S232
Abstracts of the 1996 European Aerosol Conference
degradation was studied. Samples of protein solutions were withdrawn from the liquid reservoir at different times and analyzed as previously described. RESULTS Importance of aerosolization parameters The results show that the most important parameters for the degradation of urease is the liquid flow rate, followed by the relative humidity of the atmosphere (Fangmark and Carpin, 1996). Degradation occurred both during atomization with compressed air and with N2, showing that oxidation is not a primary degradation pathway. Within the experimental range, 1- 9 recirculations, the number of recirculations was found to be the dominating parameter for the degradation of IgG, followed by the relative humidity of the atmosphere and the liquid feed rate. Thus deactivation is likely to occur at the airlliquid interface where shear forces and water evaporation may act to bring reactants closer together and produce a local environment with a high ion strength. The results also imply that denaturation is caused by aggregation. Time course of degradation Two types of deactivation behavior were observed with a Collison nebulizer. The first type, represented by urease, is a linear degradation with time on a logarithmic scale (Type I). The other type, represented by IgG has a rapid initial degradation rate that levels-off after approximately 5 - 10 minutes of nebulization (Type II). Accordingly an equal fraction of urease is degraded by surface forces during each passage through the nebulizer. For IgG, the time course of degradation correlates well with the temperature decrease of the nebulizer solution. Thus, for IgG, effects caused by the evaporation of water may be the most important. In earlier studies on the time course of protein degradation, Type I degradation was observed for the enzyme lactase-dehydrogenase while the protein recombinant granulocyte colony stimulating factor follow type II degradation (Niven et al, 1994).
ACKNOWLEDGEMENTS Support from an ERDEC-National Research Council Research Associateship award is gratefully acknowledged. REFERENCES Fangmark I., and Carpin, lC. Stability of proteins during aerosolization. The effect of operating conditions on urease enzymatic activity. Submitted to AerosolSci Techn. May, K.R. (1973). The Collison nebulizer: Description, Performance and Application.
Aerosol Sci, 4,235 - 243. Niven, R. W, Ip, A. Y, Mittelman, S., Farrar, C., Arakawa, T, and S. J. Prestrelski. (1994) . Protein nebulization: I. Stability of lactate dehydrogenase and recombinant granulocytecolony stimulating factor to air-jet nebulization. Int J Pharm 109, 17 - 26.